The long-term objective of our research is to understand the mechanisms by which obesity, and specifically upper body obesity, causes insulin resistance and the other health problems such as Type 2 Diabetes. Our focus has been on regional adipose tissue lipolysis and fat storage, as well as FFA released as a result of lipolysis affect the functioning of other tissues (lipotoxicity). The specific aims of this proposa are to: 1) assess the contribution of plasma FFA and muscle FFA uptake to the intramyocellular lipid metabolites (ceramides and diacylglycerols - DG) most closely linked to impaired activation of insulin signaling pathways in muscle and reduced glucose uptake; 2) define the improvements in muscle FFA uptake and FFA incorporation into intramyocellular lipid metabolites associated with improved activation of insulin signaling pathways and muscle glucose uptake following overnight suppression of FFA with intravenous niacin in UBO and T2DM; 3) measure the effects of sub-acute, moderate elevations of physiological FFA on muscle lipid metabolite trafficking, insulin signaling and muscle glucose uptake in healthy, non-obese adults. We will measure insulin stimulated glucose disposal (muscle glucose uptake) and muscle insulin signaling in obese humans with widely varying degrees of insulin resistance. We will also measure muscle FFA uptake as a function of FFA concentrations using 11C-palmitate PET scan approaches to understand if variations in muscle FFA uptake beyond those predicted by concentration contributes to greater lipotoxicity. We also measure muscle FFA trafficking using will use stable isotope/mass spectrometry approaches to determine how FFA are trafficked into long chain acyl-CoA, DG, TG, ceramides and long chain acyl-carnitines in muscle of these same volunteers. Our hypothesis is that trafficking of FFA into ceramides and/or DG, specifically subsarcolemmal lipids, is more closely linked with muscle insulin resistance. We will also test how lowering of FFA by an overnight, intravenous niacin infusion alters intramyocellular FFA trafficking and insulin action. Finally, by raising FFA via a duodenal infusion of palm oil vs. Intralipid(R), we will test whether a physiological elevation of FFA (as opposed to isolated increases in polyunsaturated FFA) mimics the muscle trafficking profile of insulin resistant obesity. Combined, we believe these studies will offer insights as to why some obesity phenotypes have even more variability in muscle insulin action than would be predicted based solely upon plasma FFA concentrations.